Science topic

Innate Immunity - Science topic

For those working on, or interested in, innate immunity, especially the IFN-a/b & TLR cascade.
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It seems to be an apparent consensus that the activation and function of AhR influences the landscape of TC populations, but is there a group that is more severely affected?
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T helper 17 cells (Th17) are the most affected. AhR activation may directly or indirectly also modulate the commitment of Tregs, Th1 and Th2.
Please refer to the articles attached for more information.
Best.
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As yet, no one has found the animal that gave people Covid-19. The Center for Disease Control (CDC) points out that at this time, there is no evidence that animals play a significant role in spreading SARS-CoV-2, the virus that causes COVID-19, to people.
SARS-CoV-2 is unprecedented in its combined characteristics: its long period of asymptomatic infection, high transmissibility, significant lethality in high-risk populations, being well-adapted to human cells since its emergence, and having the ability to hijack human innate immunity and bind with high affinity to the human ACE2 receptor.
The reason why we should try hard to figure out the origin of Covid-19 is to inform our efforts to prevent another pandemic like this from happening again. This one was an unfortunate and terrible accident. We should badly want to avoid a second occurrence. We can be blamed for allowing a second one like it if we do not work together soon to find the origin. Right now it appears likely it came directly or indirectly from bats. But specifics would better help us to avoid a second pandemic disaster. Furthermore time is not our friend in finding the origin and sooner is better before information is lost. We need all countries to support a real epidemiological investigation by an unbiased team of scientists given access and authority to take the investigation where ever it leads – possibly to patient zero or to the CoV-2 animal source.
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Also, have a look at this useful link.
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The THP-1 cell showed the classical monocyte (CD14++ CD16-) behavior. If we want to study the non-classical CD14-Cd16++ without drawing blood and isolate the monocyte with FACS, what can we do?
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Dear all,
I haven't found a paper yet that claims THP1 is a classical monocyte. Would you know of one? Thank you!
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 RIG-I is one of the receptors of innate immunity.
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I think, many investigators observed T cell markers on the cells of myeloid lineage (neutrophils, monocytes, dendritic cells, eosinophils, macrophages). Evidently, many of these observations can not be fully explained by artifacts due to co-aggregation with T cells. In my laboratory, we really observed TCR beta and gamma chains on 1-3% of neutrophils and CD4 on CD11c+ (dendritic) cells. In available literature, evidence for expression of rearranged chains of TCR and immunoglobulin molecules in myeloid leukemia cells can be found. My interest to these findings was stressed by recently published works by Kerstin Puellmann and Wolfgang Kaminski with co-authors, showing functionality of recombinatorial immune receptors on neutrophils and macrophages (original publication has appeared in PNAS, 2006, 103(39):14441-6.). However, I see continuation of this history still in one laboratory. My personal doubts are evoked by own practice and the reason that we do not see any support for this idea in responses to allogeneic MHC molecules. Although neutrophils can accumulate in the spleen and blood of animals, immunized with MHC class I disparate tumor cells, they cannot do this without CD8 T cells and do not contain elevated percent of TCR bearing cells. So, I am inclined to think, that T cell markers on myeloid cells represent reminders of developmental history of individual cells, deciding to change lineage too late. I would very appreciate further discussion on this issue.
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Good Question...
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I know that C57BL/6 mice are naturally resistant to most types of kidney disease models, such as the adriamycin model, however does anyone know if there are models that they are susceptible to? I know they are susceptible to the anti-GBM nephritis model, but I was just wondering if there were any others
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You can try Ischaemia re-perfusion injury model.
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Hello! I was reading papers about innate immunity and I encountered this term: "Microorganisms that invade a vertebrate host are initially recognized by the innate immune system through germline-encoded pattern-recognition receptors" ,"A set of germline-encoded receptors, referred to as pattern recognition receptors, evolved in host organisms to recognize PAMPs". But the definition on wiki (In biology and genetics, the germline in a multicellular organism is the population of its bodily cells that are so differentiated or segregated that in the usual processes of reproduction they may pass on their genetic material to the progeny) doesn't seem to fit here.
Thank you in advance.
Best regards,
David
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Yes, @Daniel McKim, I didn't mean to imply that only embryonic derived immune cells are the only ones that express PRRs. They are two separate topics. As for germline immune cells... I still consider the embryonic tissue resident APCs as germline, as most, if I'm not mistaken, are pretty homogeneous in response, unlike the bone marrow derived APCs. Although you are correct that they are more correctly referred to as embryonic derived immune cells.
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When I stained mouse PBMCs, I saw there was a population of Ly6c+CD11b- cells (>10%) and these cells express CD86. From the FSC and SSC, I can tell they are not lymphocytes. Does anybody have an idea what kind of cell type these cells are? Enclosed is a figure from a JEM (2012) paper. They could also see this Ly6c+CD11b- population.
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They seem to be common monocyte-macrophage progenitors (CD11b neg Ly6C+). I observed this population of cells as well in the spleen of mice.
Here a link to a book which explains that.
Best,
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I am trying to find the list of cells that content endosomal TLRs (TLR3,7,8 and9). Are these TLRs expressed all over the body or there are very specific cells that expresses these TLRs?
Any input is appreciated. Any reading material to find the answer is appreciated.
Thank you.
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Szczepan, thank you for replying to the question. Do you have any reading material that supports your answer? I will highly appreciate if you could share here. Thank you again.
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It seems that original wild plants are more resistant to diseases and parasites that those selected and "improved" for their commercial value
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Thank you very much Ricardo for your answer. Does it means that wild plants living in the absence of challenges will gradually lose their ability to fight against diseases and pest? could it be an epigenetic phenomenon, like the methylation of CpGs of the promoters of resistance genes?
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I am planning to identify specific innate immunity biomarkers in patients with BKC form tear samples. Can anyone advise which biomarkes are most commonly expressed (e.g. alpha-defensines, beta-defensine-2, cathelicidin (LL-37)) and what methods should I use to measure them (tear sampling)?
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This depends on the type of immune cells you expect. I would look at IL-8 and MPO for neutrophils. ECP, EDN, IL-5 for eosinophils, Granzyme B for e.g. NK cells, histamine for basophils and mast cells. To sample tears you can use schirmer strips and then you can extract the analytes.
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MNK 1 & 3 are ILC-like cell lines (Innate Lymphoid Cells) described by Allan DS et al in the article :
An in vitro model of innate lymphoid cell function and differentiation.
Mucosal Immunol. 2015
I was wondering if anyone is currently using these cells because this is the case for me and when I characterized them (stimulation- qPCR, ELISA) (Flow Cytometry), the results are just the opposite of what we're supposed to see.
Any experience or suggestions you'd like to share?
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Hello Julie,
I tried to contact the PI about the MNK cell lines, but did not get any reply. So, now I am going to try differentiating CLP into ILC3.
Vivek
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I am a fresh man in cell molecular biology. I was studying the liver metabolites function and the inflammatory response with exogenous toxicant. I knew the hepatocyte is the metabolite center for human body. It could synthesize a lot of enzymes, albumin and metabolize toxicant. But I was confused whether the hepatocyte could secrete the pro-inflammatory factor, such as TNF-α, iL-6, when liver or hepatocyte was treated with anti-cancer drug doxorubicin (DOX), and exogenous porphyromonas gingivalis. I saw two papers. They reported the upregulation of mRNA in these treatment.
[1] Zhang W, Yu J, Dong Q, et al. A mutually beneficial relationship between hepatocytes and cardiomyocytes mitigates doxorubicin-induced toxicity[J]. Toxicology letters, 2014, 227(3): 157-163.
[2]Takano M, Sugano N, Mochizuki S, et al. Hepatocytes produce tumor necrosis factor‐α and interleukin‐6 in response to Porphyromonas gingivalis[J]. Journal of periodontal research, 2012, 47(1): 89-94.
Most of other materials reported that the immune cells and adipose tissue was involved the secretion of these pro-inflammatory factor. It seems the function the hepatocyte only working on the metabolites, enzyme synthesis, and energy storage.
Thank you very much!
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Mouse mast cell lines MC/91-14 were initially described by Nabel et al, 1981, Nature, 291(5813), 332-4.
Some authors mention MC/9 cell line others mention MC/9.2 cell line. Are these two different mouse mast cell lines, being the former and latter capable to proliferate after stimulation with IL-4 and IL-10 or IL-10 alone, respectively? 
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Hi Thomas,
I have acquired MC/9 cells from ATCC and, unfortunately, according to selling contidions, I am not allow to distribute theses cells. I am sorry.
Geraldo.
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Currently, we know that HSPs can act as a danger associated molecular patter (DAMPs), under stress or immunogenic cell death conditions. When this happens, HSP70 could be released or exposed at the cell membrane, to then be recognized by the PRRs on the innate immune cell surface. But how does a citoplasmatic proteín could be translocated to the cell membrane? Is there any known mechanim that explains this? I think something in the structure of the molecule must change to get in the cell membrane, maybe it becomes more lipophilic. Does anyone knows something about this?
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Hi, is there anyone who knows about how IL-6 production is regulated by innate pathways (or other pathways) in the gut under steady state? any difference between small intestine and colon? Thanks!
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STAT 3 pathways
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Is there anyone who has information in regards to ELISA analysis done on crocodilians? Looking to assess parasite load and immune response in crocs, but I can only find one article who's tried this semi-successfully.
Is there a other/better way to measure immune response, or the lack of, in crocodiles in response to parasite load?
Cheers
Joe
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Hi Dr. Al-Salhie, unfortunately your link shows an equipment that is called "Crocodile" ( the commercial name of the instrument is crocodile, a device to pipette, wash and incubate plates only). However, Berthold technologies does not sell ELISA assays directed to crocodilian proteins, just as nobody else does for reptiles. Believe me, I searched everywhere for antibodies against snake proteins.
Thank you though, for a split second my hopes were high...
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Does it work fine? Any recommendations?
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Chemistry World has reported on this issue:
and I have a follow up at my blog as one of the two references cited in support of the SmartFlares includes plagiarism... of my blog.
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Hello, 
I am currently trying to standardise qPCR primers for IL-10 expression in human macrophages i.e. checking amplification, primer efficiencies. Before the actual stimulation by bacteria, I am using control treatments to establish the system using M-CSF mediated THP-1-derived macrophages. 
I am using the following treatments: 
1. Monocytes only
2. Differentiated but unpolarised Macrophages
3. LPS+ IFNg
4. TGFBeta1
5. Glucocorticoids mixture (GCM)
6. As a control for some experiments, PMA-mediated THP1 derived macros.
I have tested the following samples already for the expression of IL-10 and MMP12 with two separate sets of primers. 
1. Monos, 2. unpolarised macros 3. LPS + IFNg macros 4. GCM macros
Problems: 
1. I cannot detect IL-10  and MMP12 in monos (Amplified very late Ct 38 or undetermined).
2. Cannot detect IL-10 and MMP12 in M2-like (GCM treated) macros  (Amplified very late Ct 38 or undetermined).
3. Very weak MMP12 and almost no IL-10 in M1-like (LPS+IFNg) macros.
Suggestions:
1. Should I wait for the TGFbeta1 samples before concluding anything, is it usually expected in this treatment ?
2. Should I check in PMA-treated macros ? 
3. What could be a positive control for IL-10 and MMP12 ?
I would be extremely grateful if someone could show me the way here,
Cheers,
Sudip
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1. What you found is expected. I am sharing my IL-10 experience with human macrophages. Human macrophages are very different from murine macrophages. IL-10 is a murine M2 marker. It does not work in human. M2s have subtypes: M2a (IL-4), M2b (LPS+Immune complex), M2c(IL-10/TGF-b). I saw, IL-4-treated human M2s dnt express IL-10. But if unpolarized macs are stimulated with IL-10, they produce IL-10. 
2. PMA-treated macs are similar to monocyte derived macs (MDMs), but not the same. Surface marker expression, cytokine release of PMA-macs are different from MDMs. 
3. If you do qPCR, I think you need unpolarized cells as negative control/ basal expression. 
You can check this article: PMID: 25870903
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I'm having difficulty on getting an 'easy' protocol, that doesn't require many steps and reagents to get the NETs before the SEM processing. 
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Simplified suggested protocol with not so many steps.
1. Mix the standard isolated neutrophil cell suspension of fish with stimulant at a ratio 1:4. Total volume 50 microliter.  Use : 1 μg/mL of PMA final concentration as a stimulant. Both the cell suspension and PMA should have been diluted and kept in HBSS with Ca, Mg, without phenol red.
2. Shake  for 10 seconds on a shaker.
3. Incubate the suspension at room temperature for 2 hours.
4. Your NETS are now ready to be stained with whichever dye you are using and whichever protocol you are using for cell fixation.
P.S. instead of fish blood, its better to use fish anterior kidneys to isolate neutrophils.
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I am trying an antibody that binds specifically with the culturally adapted one. Giving a significant difference between positive and negative controls.
On the other hand, it does not bind specifically with the wild type in splenocytes
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Hi Kerolous 
wild type virus usually is more immunogen  than culturally adapted
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Would a good approach be to use supernatants from various breast cancer cell lines and differentiate these CD14+ monocytes from healthy donor blood into macrophages in their presence for 10-14 days? Any other insights?
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Thank you!
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NLRP3 inflammasome activation results in the release of pro-inflammatory interleukins. Several authors have demonstrated the presence of these interleukins in organs with inflammasome hyperactivation caused by intrinsic or extrinsic damage, for which kidney disease is not the exception; however, inflammasome activation has not been proved to be the cause in the light of an experimental model. Therefore, studying new therapies that focus on removing or inhibiting inflammasome components, both individually and together, is proposed in order to develop the hypothesis raised here. The involvement of inflammasome in human disease has incited efforts to identify potent and specific ways to interfere with NLRP3 activation in the context of auto-inflammatory diseases, including other diseases such as obesity, diabetes and hypertension.
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Have you seen this paper yet?
You may consider using such agent in a kidney disease experimental model.
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We want a rapid method to determine the cytotoxic potential, refering to the level of IFN-g, TNF-a, Perforin, Granzyme, without using an assay for direct cytotoxicity.
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Define "best".  FACS gives you data about individual cells - you put an antibody for each target of interest into your cells, feed it into the instrument, and then play with the output to make it look how you want, essentially.  Each cell is measured individually for each antibody, so you get a whole lot of data.  But it's comparatively expensive, as techniques go, at least if you don't have your own instrument and need to use a facility.
ELISA by comparison is pretty cheap, and can be read out on non-specialized plate readers, but tends to be a bit harder to optimize.  Once you do optimize, though, you have a fairly robust, fairly quantitative assay available.  But it only gives you bulk numbers - it tells you information about your cell population as a whole, not about individual members.  It's probably a bit slower than FACS too, unless you have robots to do everything for you and can just come back the next day for data.
Which is better really depends upon what sort of data you need, and how much you want to spend to get it.
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I tried 48 h pre-incubation with rec. m. IFN gamma (should increase IL-31RA at least in human monocyte derived DC). Then stimulation with rec. m. IL-31, but I could not detect any TNF as described for human DC. LPS leads to expected TNF "explosion".
Any ideas? My next step would be IL-6 + low (low!!) dose of LPS.
Thanks
Wolfgang
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Hi Wolfgang,
Have you considered STAT phosphorylation as read out? We are doing lots of cytokine-induced phospho-flow assays in our lab, albeit using primary human T cells mostly. We haven't tried out IFNg but type 1 IFNs result in robust STAT1 phosphorylation within minutes and is easily detectable by flow cytometry. Similarly, IL-6 induces pSTAT3 very potently in T cells (and monocytes). However, I would not think that DCs respond well to 'normal' (classical) IL-6 signaling. Although activated DCs produce a lot of IL-6, they do not express (much) of the IL-6 receptor (IL-6R) on their surface. They are, however, susceptible to IL-6 'transsignaling' using a complex of IL-6 with soluble IL-6R...still, to test your JAK inhibitors, I would probably go for type I INF (via JAK1/TYK2) or IFNg (via JAK1/JAK2) induced pSTAT1.
Hope this is useful.
Christian
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Innate and adaptive immunity
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Can you, for example, look, if LPS or PMA can activate ROS - production by macrophages?
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I plan to check the impact of exogenous cytokines in alteration of M1 or M2 phenotype using mouse macrophages. I'm not familiar with the mouse macrophage cell line. For this purpose, which cell line is suitable? I will determine phenotypes by flow (CD86, IA/IE; M1, MR; M2) and iNOS/Arginase-1 ratio by qPCR.
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The RAW 264.7 cell line is one of the best characterized cell lines representing the M1 phenotype. For the M2 phenotype, the IC-21the cell line is a good representative line(Yagnik, Landis. Arthritis Rheum. 2000 Aug;43(8):1779-89)
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wondering if anyone knows an easily available human macrophage cell line that we can use to model liver inflammation response mediated by Kuffer cell in vitro? will THP-1 or U937 line be good enough? thanks
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Hi Dear,
Please read the attached file, I think it will be helpful.
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Does anyone know what other pathogens we can use in order to derive Cell mediated immune response? or DTH?
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I have one more question Mario. Do know any paper regarding these organism that can be helpful to me. I really appreciate if you tell me the name of the paper; I probably has not seen it or you probably have some clue about a few papers regarding this . 
Thanks,
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About the natural autoantibodies in the serum of healthy individual. 
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Dear,Naael Ali
There are many different types of auto-antigens in health individual which are under suppressor manner, that when uncovered may lead to production of immune response( immune reaction ) and presence of different types of auto-antibodies (because these auto- antigens are hidden or occult  antigens with latent  bioactive functioning ). For examples, sperms (components: antisperm antibody), joints synovial fluid, ovum (components) and ocular fluids when this compartments are being (conditions) associated with trauma, infections,rupture, abrasions or any pathological causes that may lead the (body) immune system to known those hidden or covered antigens and reflect to induce an auto-immunological reactions.
Good, Luck
NASEER ALMUKHTAR 
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Is there any way to detect/measure activation of TLRs (preferably specific TLRs) in tissue sections?
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Thanks for the additional comments! Unfortunately I don't think these approaches would be specific enough, because I'm talking about tissue sections and the mentioned downstream signaling molecules are not exclusively used by TLRs, so their activation doesn't prove TLR activation.
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If mRNA would get into the endosomes of human cells would it be recognized by TLR7/8? And if it would be recognized, would it be immunostimulatory, or does the 5' cap and 3' poly A-tail prevent that reactions toward self RNA occur?
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I have tried transfection of cellular RNA into 293T cells by lipofectamine, the cellular RNA did not stimulate the cells to produce IFN. the transfection of RNA by lipofectamine may directly deliver the RNA into cytoplasm that avoid to be recognized by TLRs. 
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It is known that poly I:C stimulates mDC to produce IL12 and IL15, which are potent activators of NK cells. I am wondering if the produced IL12 or IL15 can affect pDC maturation or function. I don't get answers by google. May anyone here share their insights on this ? 
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Hi Awatif,
This paper is very helpful. The only pity is that effect of IL12 or IL15 on pDC maturation is not evaluated there.
QM
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When I use a DX5+ Miltenyi kit, the purity is between 50-60%. If I use cell sorting (without any previous separation step), the day after the sorting, NK cells are not functional for cytotoxicity assays (even if they are cultivated with IL-15 1 ng/mL). If I do first DX5+ Miltenyi kit followed by cell sorting, I lose so many cells that I can't do anything.
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Do you guys know how many Nk cells we can get per spleen using the NK cell isolation kit from Miltenyi ? thanks
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Leucine-rich repeats are present in a large number of functionally unrelated proteins. All TLR's also contain LRR but recognize different Pathogen-associated molecular patterns, or PAMPs. How does LRR play its role in TLR's and do a number or LRR have any role in function of TLR's?
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how can i identify the LRR domain of a TLR?
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In mammals, antibodies are classified into five main classes or isotypes – IgA, IgD, IgE, IgG and IgM, according to the heavy chain they contain. Lets say in an effective immunization program, and looking only IgG, IgA and IgM, IgM will first be elicited, which will then undergo isotype switching to IgG and IgA. Why we need different types on antibodies targeting an antigen? Can I say that due to the localisation of the antibodies? For example, IgG is a circulating antibody (abundant in serum) while IgA as mucosal antibody (IgA is the predominant antibody in mucosal secretions)? 
Sorry for asking about basics because I am getting confused and get deviated when I read more. I hope there is someone to explain to me a little bit about this. Thanks a lot!
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In mucosal tissues, IgA is the most abundant. IgA is transported at high levels from the serosal side to the lumen by the pIgR. Bacterial stimulation of the intestinal epithelium induces the production of IgA. However, translocation of bacteria over the intestinal epithelium results in increased induction of IgG. IgG1 and IgG3 increase following exposure to the protozoa Falciparum. Whereas IgG4 is more dominantly present in soluble antigen responses and helps to prevent or reduces IgE-mediated allergic responses.
IgD is expressed first during B cell development, followed by IgM. Although the role of IgD is still partly illusive, recently IgD has been implicated in various innate immune functions: http://www.nature.com/ni/journal/v10/n8/abs/ni.1748.html.
The biology of the regulation of IgA1 and IgA2 is intriguing. Both are secreted equally efficiently by pIgR. However, as mentioned above, levels could vary between tissue suggesting differential regulation of their respective expression and CSR mechanisms. The innate factor APRIL seems to advantage the production of IgA2 over IgA1, which correlates with increased expression of IgA2 in the bacteria-rich colon: http://www.sciencedirect.com/science/article/pii/S1074761307002877.
There are numerous reviews out there, but one last comment about membrane-bound versus secreted Ig: These versions both occur for all isotype classes and are independent of CSR, but do depend on alternative splicing, where the first heavy chain domain is required for membrane-bound Ig (also referred to is BCR) and the cleaved form allows for secretion of Igs as predominantly observed in (terminally) differentiated plasma blasts and plasma cells.
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How I can block NKG2A receptor signaling for in vitro experiments?
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You might consider to convert your anti-NKG2A antibody of choice (and the respective isotype control) into Fab/F(ab)2-fragments - there are several commercial kits for that purpose that are easy to use and reliable. By using blocking Fab/F(ab)2-fragments you circumvent the potential problem of Fc-receptor-mediated activation. Good luck!
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We've been looking a kit to measure the amount of endotoxin in mouse serum (with high-fat diet or not). What's the most reliable and practical kit for this? I found a thread about this but it has been a year. We also looked into the papers and some used QCL-1000 from LONZA. Another new kit is EndoLISA. Does anybody have hands-on experience on any of these? Thanks for your suggestions.
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we have hands-on experience on measure  the amount of endotoxin in mouse serum
we used the following kit.it was good
Mouse Lipopolysaccharides(LPS) ELISA Kit cat.NO: CSB-E13066m
supplied by Genxbio Health Sciences Pvt. Ltd.
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I am aware of RNAi-based therapeutics but am not clear about the natural response of the human body to viruses via RNAi. I remember some article that said RNAi is active at early stages of life and is then slowly replaced by protein-based immunity. I cannot find that paper. I would greatly appreciate a PDF or link to that article or a similar one.
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Yes the RNA based interference is existed in human. the newly article publish recently have uncover a group of family existed in eukaryotes and human doing RNA interference . you can find the articles under the title "DNA-guided genome editing using the Natronobacterium gregoryi Argonaute". this technique is more powerful than CRISPER-Cas9, just their plasmid has been not introduce yet. 
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Does anybody has the protocol or information how I can optimize dose of antigen in PBMC culture? How the results should be addressed? Is cell viability result is enough or I should test gene expression level?? My research target is to investigate gene expression. If upto gene expression is necessary, which innate immune genes may be tested??
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In order to optimize the dose of antigen you could titrate your antigen and quantify a cytokine like IFNg or IL2 using an ELISA kit. You could access, in paralel, cell proliferation.
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Hello all,
I'd like to detect pyroptosis and necroptosis in tissue such as skin and skin-draining lymph nodes.
I guess that target cells are most likely epithelial cells or innate immune cells, so isolation and flow cytometric analysis of these cells might not the best way for these cells.
Therefore, I'd like to find ways to probe these types of cell death in tissues. Can you guys recommend me what would be the best markers for these types of cell death, and which analysis (histology, IF and others?) would be the best?
Thank you!
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For pyroptosis you should canonically look for activation of caspase-1 by western blot (the p10 or p20 fragments; use the antibodies SC-514 [mouse] or SC-515 [human] from Santa Cruz Biotechnology or Casper-1 from Adipogen). You may also look for the perinuclear inflammasome foci usually associated with pyroptosis that can be detected by immunofluorescence using antibodies or with the non-antibody YVAD-FLICA tag. An example can be found in this recently published paper: 
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Dear all, 
Your kind advise is needed here: I am working with mice- in which we initiate a trauma for inflammation activation. I am planning to block one of the anti-inflammatory cytokines' receptors. My Questions is: Is the activation of the innate immunity after a trauma goes in parallel with the anti-inflammatory cytokines activation? Or in other words, what is the best time to block one of the anti-inflammatory cytokines' receptor after initiating the trauma?
Am thankful already :) 
With my Best Reagrds, Alaa
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Generally, the immune response after trauma, infection, sepsis, starts first with the activation of Th1 immunity and proinflammatory cytokine production (SIRS). As our body knows, how dangereous for our  organs can an uncontrolled hyperinflammation be (multiorgan dysfunction syndrom, multiorgan failure, failure of microcirculation, death due to cytokine storm even in the absence of microorganisms), approximately 10-24 hours later starts with the compensatory antiinflammatory response (CARS)) -  with the production of Th2 (IL-4, ...) cytokines and production of antiinflammatory cytokines (IL-10). The  aim of this is to perform a balance. If this compensatory antagonistic response is too strong, the Th1 immunity together with inflammation is strongly inhibited, and it can lead to immune depression even immune paralysis that might be the cause of development of e.g. polymicrobial sepsis. Note: the higher of hyperinflammation is, the higher the compensatory antiinflammatory response will be.
Cave: Patients with head trauma or patients suffering from immune deficiency can from the beginning start with Th2 immunity and CARS, that means without hyperinflammation and Th1 immunity actovation.
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Endotoxin can possibly be neutralized in vivo by diverse substances. The endotoxin- neutalizing substance complex must be removed from the body before it could cause CD14-TLR4-mediated activation of leukocytes and release of inflammatory cytokines leading to ill-effects of endotoxemia. A single mechanism or it will depend on the type of the complex formed?
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Hi Gaurav,
Thanks for the answer. In fact, I was aware of this system (a sort of hemodialysis) to remove endotoxin by polymyxin.  Rather I was curious to know about the fate of endotoxin trapped by its binder in vivo. If we consider reticuloendothelial system, it is likely to invoke innate immune system in somewhat altered manner (LPS in the endosomal compartment also detected by innate PRRs, until and unless it is degraded by the enzymatic system therein).
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I am working with macrophage cells and time is of the essence with what I am trying to do. I know that inflammasome formation requires "two hits" of some type of immune stimuli in order to form and for the subsequent secretion of IL-1B, IL-18, and other cytokines. Many people use LPS and ATP for inflammasome formation, but I was curious as to whether or not it was possible to stimulate inflammasome formation in the cells with the "two hits" simultaneously i.e. make a solution of both LPS+ATP. Additionally, what seem to be the best concentrations for both stimuli? 
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The best answer is "probably" yes. Your readout will be the deciding factor. If you are looking for:
  • Specks by microscopy
  • IL-18 release by ELISA/WB
  • Caspase-1 cleavage by WB
  • ASC dimerization by WB
Then yes, it would probably work. The following paper showed that licensing of the inflammasome is rapid and only requires on the order of 5 minutes to engage:
Effectively, the inflammasome requires post-translational licensing. The long duration priming that most people use is usually to synthesize IL-1b and additional amounts of components such as NLRP3. However, NLRP3 is present in most cells at a low level constitutively and IL-18 is definitely constitutive. As such, a brief priming with LPS or some other "signal 1" stimulus will be sufficient for NLRP3 inflammasome priming.
Using them together would probably have the intended effect, but you run the risk of the P2X7 engagement with ATP throwing the system off of balance since it is detected and responded to within a few 10s of milliseconds. It's best to do a one-two punch with a brief 5-minute priming phase followed by ATP.
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Hi, the attached contain a photo of stained mussels hemocytes with MGG, could someone help to annotate those cell types, the colors are very close, so I don't know if there is a good colours or not? thanks in advance
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Dear Younes,
Most haemocytes in your figure are granulocytes (arrows in the Fig. that I am attaching). In those granulocytes the central area appears filled with acidophilic granules, which hide the nucleus, and the peripheral area is hyaline. Although you do not mention the procedure, I guess you have deposited a drop of haemolymph onto the slide and allowed the haemocytes to adhere spontaneously onto the slide for a while; by this way, granulocytes spread on the slide surface and adhere onto the slide, thus resulting in this appearance with a peripheral hyaline area (ectosplasm) and a central area (endoplasm) filled with granules. There are also some smaller haemocytes (double arrow in the Fig. I am attaching), which do not show almost any cytoplasm, with a patent nucleus; this smaller haemocytes could be hyalinocytes or undifferentiated stem-like cells. With the procedure of spontaneous adhesion onto the slide, hyalinocytes and stem cells are mostly lost because they have much lower ability to spread and adhere than granulocytes; this is because those smaller haemocytes show reduced or almost null cytoplasm.
If you want to keep all the cell types in the slide you could either cytocentrifuge haemolymph or to use slides covered with an adhesive film; slides covered with poly-L-Lysine are frequently used. You can contact me for further explanation or any other information on this issue. I am also attaching a paper on mussel haemocyte morphology. Please, confirm that you have read this message. Best wishes,
Antonio
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Please, advise me any publications in this topic would also be helpful. 
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just because they are close does not make them the same!  The endometrium is a completely different environment and I would say not influenced by what is going on in the cervical region 
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I know MCSFR (CD115 or CSF1R) mRNA is expressed by GMP (granulocyte-macrophage progenitor) and CMP (common-myeloid progenitor), but what about protein? Is CD115 detectable on the cell surface of GMP and CMP?
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This work might help!
www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)
by YK Lieu - ‎2012 
Impaired adult myeloid progenitor CMP and GMP cell function in conditional .... No abnormality was detected in the pIpC-treated mybf/+/MxCre mice (data not shown). ... Cell surface expression of CD11b, CD41 (a marker of megakaryocytic ... in the CD11b+Gr-1- compartment also expressed elevated surface CD115 and ...
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I was working on C-type lectin-like receptors of the Dectin-1 cluster, especially on CLEC-1, during my PhD project. We tried to find the ligand, but although we tested everything we could get hold of, we couldn't find it.
It seems to me, others might have encountered similar issues. One by one all the other receptors in the family have been characterized thoroughly and there are lots and lots of papers on Dectin-1, CLEC-2, LOX-1, CLEC9a and so on. However, hardly anything on CLEC-1.
I'm working on something very different now, but CLEC-1 is still haunting me...
Is anybody out there still working on it, so that I might find out eventually, what it actually does?
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Hi Elise, sorry for the late reply! I'm happy to hear that! I haven't been working on CLEC-1 anymore for a long time. CLEC-1 was part of my PhD project and I was looking for a ligand but couldn't find one. I'm still very curious about it though...
We've published our CLEC-1 data in 2012 (Scand J Immunol. 2012 Mar;75(3):282-92. The human C-type lectin-like receptor CLEC-1 is upregulated by TGF-β and primarily localized in the endoplasmic membrane compartment.) We didn't find a ligand. However our approach was not very systematic, instead we tried whatever we could get our hands on for free. This is what we say in the paper: 'Potential candidates tested were known ligands of other members of the myeloid subfamily of C-type lectin-like receptors (e.g. zymosan, oxLDL and podoplanin), intracellular pathogens (e.g. Toxoplasma gondii and Listeria monocytogenes), ligands of intracellular Toll-like receptors (e.g. the TLR9 ligand CpG) and several primary cell types and cell lines.'
I wish you all the best with your CLEC-1 project!
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I am interested in the phenotype (very reduced neutrophils and macrophages populations) at 4-5 dpf and was wondering if the morpholinos were efficient that late and if the fish development was normal.
Original paper: Li J, Li K, Dong X, Liang D, Zhao Q (2014) Ncor1 and Ncor2 play essential but distinct roles in zebrafish primitive myelopoiesis. Dev Dyn 243:1544–1553
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@Namita, thank you very much for your answer.
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Innate lymphoid cells (ILCs) are a group of innate immune cells that belong to the lymphoid lineage but do not respond in an antigen-specific manner, as they lack a B or T cell receptor.This relatively newly described group of cells has different physiological functions, some of them analogous to helper T cells, while also including the cytotoxic NK cells. In accordance, they have an important role in protective immunity and the regulation of homeostasis and inflammation, so their dysregulation can lead to immune pathology such as allergy and autoimmune disease.
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It's worth pointing out in case you didn't realise that Hergen Spits is the foremost authority on human ILC. To add to his ST2 comment: I've only tried the MBL anti-human ST2 (clone 2A5) and it was nothing like I'd seen with the mouse. I would very much like to know what a good anti-human ST2 clone is.
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During persistent chlamydial infection innate immunity also plays very important role, macrophage polarization is supposed to be one of the reason, how to differentiate this polarization efficiently in-vivo ?
as pathogenesis of chlamydia in reactive arthritis is not clear and it may answer few question regarding this
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Dear Kumar,
You do not explain whether you want study chlamydia infection in humans or in animal models. However, even for animal models, complete in vivo studies are hard to develop. Generally, to analyze macrophage polarization, you should immunophenotype macrophages in situ (skin biopsy) using flow cytometry or immunofluorescence microscopy . To do this, you stain macrophages with different antibodies against specific markers . Based on their differential expression, you can establish the frequency of each subpopulation (M1/ M2) and the absolute number.
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There's an interaction with a protein, FAP2, on a bacteria, F. Nucleatum, with TIGIT on natural killer cells. I want to target the natural killer cells specifically to block this interaction between the bacteria and the NK cells. I wanted to insert an antibody in mice transgenic for human TIGIT, however if I do so this will elicit an auto-immune response. How can I get around this? I'll consider alternative approaches outside of targeting FAP2 and/or the bacteria.
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You can make F(ab')2 fragments of your antibodies - there are lots of protocols for this available, but the most common way to do it is add pepsin for a few hours to cleave the Fc portion of the antibody. This will still block the receptor, but without the Fc portion it won't activate complement or Fc-receptor-mediated responses. This assumes that your antibody is a blocking antibody, and if you are not sure of this then you should test it in vitro first.
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I am currently working with human NK cell cytotoxicity. We found high expression of perforin and granzyme even in untreated pure NK cell group. It seems like the high expression of perforin and granzymeB have nothing to do with cytotoxicity. I think the trend of CD107a expression must correspond to perforin and granzymeB. But we  found CD107a  corresponding to the increase of cytotoxicity.  
Any suggestion? Any other factors are related to CD107a expression and cytotoxicity?
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I don't find this surprising, the human NK cells will simply contain a high amount of perforin and granzyme B to begin with.  The activation status of NK cells may be a better indicator of actual cytolytic capacity at a given time if perforin/granzyme B levels are similar.  Think of NK cells as a loaded gun, but pulling the trigger requires the right balance between activating and inhibitory signals.
CD107a expression is commonly used to indicate degranulation and therefore should correspond to cytolytic capacity (since this relates to release of lytic granules such as perforin and granzyme B).  If anything, the protein amount of perforin and granzyme B per cell may slightly increase following activation or stimulation.
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Integrated blood ROS generation is too time consuming, preferable could be one time point in the begin of blood ROS generation.
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Dear Thomas,
Thanks for asking this very important question.  You are absolutely right.   PMN are the primary mediators of the rapid host defense against most bacterial and fungal pathogens.  Cancer chemotherapy or reactions to cytotoxic drugs are known to cause iatrogenic neutropenias leading to severe immune deficiency.  Activation of neutrophils and to enable their phagocytic properties requires increased oxygen consumption of molecular oxygen.  The consumed oxygen is then utilized by NADPH oxidase generating ROS.  Some of the suitable methods to quantify ROS generation are as follows:
1.  Chemilunminescence amplified by luminol or isoluminol
2.  the absorbance change following reduction of cytochrome c
3.  The fluorescence increase upon oxidation of PHPA (p-Hydroxyphenyacetate).
I will be happy to provide you with the details of these techniques should you be interested.
Best Wishes,
Omer
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Hi, everyone.
I`m trying to activate NLRP3 inflammasome in various cell types with classical method (LPS + ATP).
While I reviewed this process, I noticed that the concentration of ATP I`m using (2mM or 5mM, which is considered as normal to induce NLRP3 inflammasome activation in many papers) is much higher than the concentration that is known to be cytotoxic (less than 100uM...).
I cannot find any difference in viability between control and LPS+ATP treated cells.
How is it possible? Is it LPS priming that protect cells from ATP-induced cytotoxicity? Or Do I misunderstand the inflammasome activation process/ ATP-induced cell damage? Please help me.
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How long is the ATP added for and how are you preparing your ATP solutions? What organism is your LPS from? You can do a western blot for intracellular pro-IL1b induction to determine if your priming step is the issue. I do 1 ug/mL for 4 hours and get robust induction as determined by both WB and IF.
I would recommend at least 30-60 minutes of ATP exposure. You can use a membrane-impermeant dye like ethidium bromide to determine if the P2X7 receptors are being activated by the ATP. I would recommend doing immunofluorescence for ASC or Caspase-1 or YVAD-FLICA to see if you have perinuclear specks that are indicative of inflammasomes.
5 mM ATP for 30-60 minutes will kill your cells, for sure. You can use a caspase-1 inhibitor like YVAD-CHO or YVAD-CMK and if you see a reduction in death then you are good to go.
Westerns on concentrated supernatants for IL1b or Caspase-1 are also a sure-sign, or ELISA for IL1b.
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I am testing inflammatory properties of a marine sponge extract. this extract gave anti inflammatory activity in first 2 hours with respect to the low doses in carrageenin induced paw oedema test and anti inflammatory activity for leukocyte migration in vivo test (for peritoneal macrophages)using mice, for all the doses. Can this be explainable? Can these two properties present in the same compound (this is an extract which is not fractionated)?
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You have a mixed extract with potentially 100s of biologically active compounds.  Thus, this is almost to be expected.  Further purification is indicated. 
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I need to transfect RAW cell lines and I need a good transfection protocol that doesn't include viruses or electroporation.
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Hi Noha,
for transfection RAW macrophages I use a new transfection reagent called Viromer.
Its a synthetic polymer zero in charge. Due to an active endosome escape mechanism I get higher transfection efficiency compared to standard reagents.
Please find attached some exemplary data.
Feel free to contact me in case of question: Olivia.Zabel@lipocalyx.de
Best, Olivia
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I want to stain mainly the neutrophils and monocytes from the fresh blood. I am looking for any bead based product or any that persevere the blood sample for longer time before stain the innate immune cells.
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Hi Srinivas, the only way to get reliable and robust data from neutrophils is to analyse them almost immediately after the sample has been taken.  
At 6 hours ~30-50% of the neutrophils in a sample will be dead, and by 24 hours, 70-90% will be dead (dead=apoptotic).  The best advice I can give you is that you should stain your cells as soon as you are able, and then analyse them immediately after that.  There is nothing available that will preserve neutrophils in an unaltered state for more than a couple of hours (significant phenotypic changes occur in whole blood neutrophils incubated for even 4 hours ex vivo), so you will not be able to obtain data that are relevant to the in vivo situation by delaying your sample processing and analysis.
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For example coated bateria with serum and uncaoted bacteria, what is the difference between these two in terms of phagocytosis?
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In serum the immunoglobulin G and complement proteins  can improve the phagocytosis of bactéria.
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Hello all:
I am interested in isolating pig monocytes from whole blood. I will do so by Ficoll followed by MACS for CD14 marker. 
My 2 questions are: does anyone know the best culture media for maintaining/proliferating adult monocytes, even if not specific for pigs? Also, I want to co-culture these monocytes with plasma I obtained before (kept at -80 since sampling) and evaluate expression of HLA-DR by flow cytometry. Does anyone have an idea of the time I should let the monocytes be co-cultured with the plasma? Should I do step-wise increases in the percentage (vol/vol) of plasma I should add to the culture media prior to deciding how much to add? I dont have a lot of plasma sample from each of the studied animals, so I really need to be cautious with how much I use. I am thinking of using 96 well microplates for culturing so I need little volume from plasmas for the co-cultures. 
Any advise will be greatly appreciated.
Thanks a lot.
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Hi,
Pig monocytes do represent a quit big group of cells which are characterized by different types of surface markers including CD14. Subgroups of monocytes including antigen-presenting cells exert different functions. I am wondering about the purpose of your cell isolation ?
Have a nice weekend !
Regards,
andrea
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I am looking for any article highlighting differences between peripheral and central (CNS) inflammatory reaction. With main focus on the mediators.
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I believe you can find useful information in references
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What is the difference between ADCC and opsonization?
Both produces phagocytosis by antibody receptor.
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Antibody dependent cellular cytotoxicity ADCC                                                    www.youtube.com/watch?v=bqD-bwJemlY                                                                        Antibody-dependent cellular cytotoxicity (ADC) is a tripartite immune response whereby cytophilic antibody (Ab) directs NK cells to lyse sensitized targets. In this scheme, Ab may bind to Fc receptors on NK cells which then recognize targets specific to the Fab portions of such Ab; alternatively, cytophilic Ab bound to targets are secondarily recognized and lysed by NK cells. ADC has been demonstrated to be a potent antiviral cytotoxic mechanism both in vitro and in vivo against common human viruses including herpes simplex (HSV), measles, Epstein-Bar (EBV), and influenza virus. In addition, ADC responses have been demonstrated to play a role in the control of retroviral infections and retrovirus-induced tumors in animal models.                                                                                       Opsonization: Opsonization is the process by which a foreign particle, particularly a microbe, is coated with plasma proteins (opsonins) so as to facilitate the attachment and internalization of that particle by a professional phagocytic cell. In general, the process refers to coating of the microbe with immunoglobulin molecules (antibodies) that are specific for antigenic determinants on that organism, or with 3 complement proteins (particularly C3b) deposited on the surface of the organism via either the classical or alternative activation pathways.
The presence of these plasma proteins on the surface of the microbe facilitates their sequential interaction with 3 immunoglobulin receptors (Fc receptors) or complement receptors (CR) on the phagocyte surface. These interactions result in encirclement of the particle by the cytoplasmic membrane of the phagocytic cell, until the particle is contained within a membrane-bound vacuole (phagosome) within the cell.
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If mice are given one single dose (0.5mg) of anti-Ly6G (1a8) IP to deplete neutrophils, how long will it take for the neutrophils to be replenished to near normal levels?
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Dear Evan,
We tested this using 0.1 mg 1A8 (BioXCell) i. p. (Day 0). We saw an approx. 80% depletion which lasted until Day 5. Neutrophil numbers started to increase on Day 6 and returned to normal on Day 7. Higher doses of 1A8 did not cause much more severe depletion in our hands but may last a few days longer (we did not test it). You should be careful about how to stain for neutrophils in the presence of depleting doses of 1A8 since the large circulating 1A8 concentration blocks the binding of additional (e. g. fluorescently labeled) 1A8 molecules to the epitope. We used an Ly6B (7/4) antibody to label neutrophils and gated out monocytes based on FSc/SSc. The 7/4 mAb works fine even if neutrophils are saturated with 1A8. You may also consider using the NIMP-R14 mAb for neutrophil depletion. It gave us much more robust (practically complete) depletion which lasted for 3-5 days. The specificity also seemed to be OK. You can look up Weber et al., J Exp Med 2015 (in press; link below) for further details.
Best wishes,
Attila
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Are there any inhibitors for complement receptor 3 that can be used for in vitro studies?
Thanks
Aled
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Depends on what kind of inhibitor you want to use.
There is a number of mAbs that inhibit either the ligand-binding domain of CR3, such as CBRM1/2 or bind to the beta I domain and block the relay of allostery (TS1/18 for example).
If you are looking for a small molecule inhibitor, Roche has developed some. Namely XVA143. And I think Genentech has 2 similar compounds.
Otherwise, CR3 binding is metal-ion dependent, so if you just add EDTA, you should abolish its function. 
Hope this helps.
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lactoferrin is used as immunostimulant
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A Google search on "Bovine lactoferrin antioxidant pdf returned about 2,14,000 hits
Antioxidant Effects of Bovine Lactoferrin on Dexamethasone ...
first page dump:
by L Safaeian - ‎2014 - ‎Related articles
Nov 19, 2013 - International Scholarly Research Notices is a peer-reviewed, open access journal covering a wide range of subjects in science, technology, ...
Missing: search
Effect of selenium-saturated bovine lactoferrin (Se-bLF) - DRO
by H Burrow - ‎2011 - ‎Cited by 6 - ‎Related articles
Apr 5, 2012 - bovine lactoferrin ... The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), ... Search Google Scholar.
Antioxidant enzyme activities of iron-saturated bovine ... - DRO
by H Burrow - ‎2011 - ‎Cited by 17 - ‎Related articles
Dec 5, 2011 - Antioxidant enzyme activities of iron-saturated bovine lactoferrin (Fe-bLf) in human gut epithelial cells under ... Search Google Scholar.
Patent CA2141961C - Antioxidant - Google Patents
The present invention provides a safe antioxidant applicable for food, drug medicine, non-medical drug and other products. ... Advanced Patent Search .... Kogyo Campany), bovine lactoferrin (manufactured by Sigma Company), hydrolysate of ...
Patent US7326775 - purification of transferrins ... - Google
Feb 5, 2008 - Advanced Patent Search ... 3) purifying said antioxidant-treated lactoferrin with at least one polyphenol to form purified lactoferrin; and .... Oral administration of bovine LF (40 mg/day) in healthy human volunteers (n=17) ...
lactoferrin 339615-76-8 - The Good Scents Company
Food Additive : Functional use(s) - antioxidants. ... Scientific Opinion on bovine lactoferrin:page or pdf ... Google Scholar : Search, Google Books : Search.
Lactoferrin Cancer Miracle- Super Immune-Booster - percys ...
Dec 12, 2014 - Your ads will be inserted here by. Google Adsense. .... antioxidant activity of an oral supplementation of bovine lactoferrin in humans. Using an
Lactoferrin - Advanced Health and Life Extension
Lactoferrin boosts immune function and has antioxidant properties. ... Advanced Health & Life Extension. Google. Custom Search ... The lactoferrin concentration in bovine (cows) milk is only 0.5% to 1.0% while human breast milk can contain ...
Full Text - Poultry Science - Oxford Journals
by L Wang - ‎2008 - ‎Cited by 24 - ‎Related articles
Institution: Google Indexer; Sign In as Personal Subscriber ..... (2008) found the similar effect of bovine lactoferrin, an antioxidant, can improve antioxidant ...
Patent US4668771 - Method for separating bovine ... - Google
Advanced Patent Search ... Method for separating bovine lactoferrin from cow's milk and purifying same ... A method as claimed in claim 1 wherein the bovine lactoferrin adsorbed to the ..... US7326775, 2 Dec 2005, 5 Feb 2008, En-N-Tech, Inc. purification of transferrins using surfactants, antioxidants and flavonoids, than ...
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I'm evaluating bioactivity of IL12 on different mouse strains splenocytes (C3H/Hej, C57BL/6J, BALB/cByJ). I check IFN gamma production for this evaluation. The results showed that C3H/Hej secretes a ton of IFN gamma, whereas the rest mouse strains didn't do that.
Does anyone know the reason for that phenomena?
Thank you so much for your support!
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Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows
significant potential as a vaccine adjuvant and anti-cancer therapeutic.Different cell types and soluble factors are involved in the induction of IFN-γ. Thus, in addition to IFN-γ-producing cells (T cells, NK cells, and macrophages) and accessory cells (macrophages and dendritic cells) a number of cytokines are known to participate directly or indirectly in the induction of IFN-γ. Among the soluble factors, IL-12 is of special interest, being capable of directly inducing IFN-γ in T and NK cells and promoting T cell differentiation in the IFN-γ-producing Th1 subset. Splenocyte suspensions is prepared from spleens of 6- to 8-wk-old mice by pressing spleens through a wire grid. Pooled cells from three or four animals are  suspended in serum-free DMEM, adjust to a concentration of 107/ml, and place (2 × 106/well) in 96-well plates (Nunc, Roskilde, Denmark). They are then cultured in the presence or the absence of stimulating agents (10 μl/well) at 37°C in a humidified atmosphere containing 8% CO2 for 24 h. Culture supernatants for determination of IFN-γ are stored in aliquots at −80°C until use.IFN-γ in supernatants of splenocyte cultures and in murine plasma is estimated by  ELISA. The limit of IFN-γ detection is 60 pg/ml. IL-12 (p70) in supernatants of macrophage cultures is estimated by ELISA using anti-IL-12 mAb (C17.8) and biotin-labeled anti-IL-12 mAb (C15.6; PharMingen, Hamburg, Germany) as described previously (45). The limit of detection is15 pg/ml.
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I want to know about the polymorphism in DDX,H1F1and LP2 genes during the virus sensing in a host innate immunity.
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RIG-I-like receptors (RLRs)  cytoplasmic sensors of pathogen-associated molecular patterns (PAMPs) within viral RNA. play a major role in pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity.
The RLRs detect viral RNA ligands or processed self RNA in the cytoplasm to triggers innate immunity and inflammation and to impart gene expression that serves to control infection.RLRs cooperate in signaling crosstalk networks with Toll-like receptors and other factors to impart innate immunity and to modulate the adaptive immune response
patients with single nucleotide polymorphisms (SNPs) in genes(RIG-I, MDA5, and LGP2 )encoding become highly susceptible to RNA virus infection  
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PolyIC has been used extensively in in vitro experiments to mimic viral infection in cell lines. Can anyone please refer me to an article or publication/document showing that polyIC can be used in vivo (inoculation or injection, etc) to activate antiviral innate immunity. 
Highly appreciated
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There are numerous studies to demonstrate the antiviral response that is induced by poly I:C in mice (following an injection). It is through induction of type I interferons (IFNs), which through induction of the IFN-activatable genes exert the antiviral effects. Please see papers beginning 1975.
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I know that macrophages reside in most tissues/organs, but I am wondering if anyone is aware of tissues or organs that have relatively high levels of macrophages (in healthy tissue, not during inflammation or other disease)?
For instance, the synovium of the joint has a fairly high percentage of macrophages.  
Any other examples?
Thanks!
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I wonder whether Jonathan really  had asked us  about which orпan/tissue from one can best get macrophages for culturing (as most of answerers  evidently understood him) or about organs in which a significant population of resident (sic!) macrophages plays  an important functional role. I believe these are two different questions. and answers are not the same.  It is rather easy to get macrophages for in vitro experiments from peritoneum by washing it (even without preliminary activation) but I am not sure that  their role in situ is great1). On the contrary, the red pulp of the spleen would not be able to catch senescent erythrocytes from the blood for utilisation if it were not rich in resident macropgages, but if you look simply for a convenient source of macropgahes for culture,  the spleen or the liver (in which Kupfer cells also have a very important function) would not be the best choice.
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1) Moreover, I am not sure that peritoneal macrophages meet definition of the resident ones as a tissue-specific poulation of macrophages self-maintaining locally  with only  minimal contribution from circulating monocytes.
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Hi all,
I've got small issue with isolation of epithelial cells (enterocytes or progenitor cells) from bovine small intestine.
I use chelation method (EDTA+DTT), receive crypts and seed them in rat tail  collagen coated flask with DMEM (high glucose) supplemented with EGF, NEAA, T3, hydrocortisone, insulin and 5% FBS.
Next day I can see attached cells around the crypts (island pattern) which is really good. Later on they grow really slow and after 3rd day they detached and died.
What's wrong with my method which is based on another papers?
I'll be really thankful for any suggestions.
Cheers
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Many thanks Kelli for all advices and papers.
I need a pure population of epithelial cells, not organoids, for co-culture model with fibroblasts. So I would like to grow them around 2 weeks.
Btw. Congratulation of really good paper.
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I have had experience implanting estrogen pellets (from Innovative Research of America) in nod-scid mice and at week 4 onwards, some form of toxicity and mortality can be observed. My experiments involve MCF-7 xenografts. I am moving on to NSG mice as they are better models for metastasis....hence I am wondering if the same problems (bladder stones etc) still apply to NSG mice.
Also, I have ever read a paper mentioning that estrogen supplementation is not necessary for MCF-7 growth in NSG mice... anyone can confirm this?
Thank you very much in advance!
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Supraphysiological levels of E2 can provoke anorexia, so keep an eye on food intake and weight loss.
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I have been trying to measure mouse plasma/serum IL-12 by multiplex TH1/TH2 cytokine kit (MSD). And the IL12 concentration from healthy mouse is around 1ng/mL from my measurement. For other cytokines, such as IFN-γ and TNF, they cannot be detected. I tried the IL12p40/p70 ELISA, and it gave me similar result. I am wondering whether this concentration of IL-12 is normal. 
Thanks!
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I suggest measure IL-p70 and not p40.
Good luck.
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Some research studies clearly establish that the elevation of hs-CRP is associated with increased cardiovascular risk, while others fail to find this relationship. Where is the problem of this disagreement?: in the populations studied,in the design of the studies, or in the method of measurement?
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Hallo,
First of all it depends on the standards used. The WHO (Fa. Behring) has produced 50000 liters of pure standard CRP. If this standard is used, then the first problem is solved. Then, there is the reproducibility of different labs. If you take the Ringversuche of DGKL for glucose, for example, which is so well defined almost as no other molecule!. The target value of one was 100 mg/100 ml. The values offered by well known german laboratiries varied between 80 and 180 mg/dl. Now you have the answer!
greetings
Sighart Golf
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Having read this review which I think is perfect, but I am not recognized as immunologist to make such estimations, I am even more convinced that late "Charly" Janeway was a real pioneer. I happily remember his visit at Stanford when he gave a talk in Irv Weissman's house with a huge dinner, a thrilling experience with so fascinating views and scientific as well as social anecdotes. But I guess, we should not only judge good scientist from the awards they received or not.
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Yes, indeed. It is quite often very difficult to prove a hypothesis or to find some more hints to a theory and against the paradigms.
It took decades to find some experimental support for some parts of Einstein´s theories, or, more recently, for the Higgs boson.
And there often are more than just one, two or three people involved in great findings. In the 1990s an immunologist in Zürich got the Nobel prize, but I am sure many expected Hugh McDevitt to get it as well since his findings were earlier. A few years later, McDevitt got a different award - and I think it was Irv Weissman who mentioned in the laudatio in a Montreal meeting that this was the "biggest award in immunology"  (it was much more money than a Nobel prize). Not sure what the former Nobel winner sitting behind me thought at that time, but it is not the recipient of an award who makes the decisions.
So, it may just remain hard to know and understand all the details which lead to the decisions of the Nobel committee.
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Is it known which cell type(s) produce 15d-PGJ2 and in response to which stimuli? I'm not very familiar with PG biology and I find the literature on this specific matter a bit confusing and contradictory. Any pointers will be highly appreciated.
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Sorry,I meant 
Dear Rune,
 
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Recently I have been working with detecting serum IL12 by ELISA. Based on that result, the sera from healthy mice have around 10000~20000pg/mL IL12p40/p70. But when I searched the literature, IL-12p40 is around 200pg/mL. The coating antibody and detection antibody I used can both recognize p40 and p70. The standard curve looked fine. I diluted the serum by 30 fold, and I could still detect the OD signal. And the negative control (buffer alone) had a low background.
I am wondering whether it is because the antibody can detect both p40 and p70. Still, the concentration of p40 plus p70 is too high. And does anybody know what specific antibody should I use to only detect IL12?
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Dear Daxing
Hakeem Sam and Mary M. Stevenson have described specific antibodies that may resolve your issues.
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Going back as far as I can remember, people have been to-and-fro about the benefit or deficit of an immune or inflammatory response in human malignant disease and/or metastatic progression. Tumour-Associated Lymphocytes (eg Rosenberg), Tumour Associated Macrophages (eg Fidler), Melanoma-Specific Antigens, CD4/CD8 ratios, Th17/Tregs, IL-10, gamma-deltas, MDSCs, Antigen-Pulsed IFN-primed DCs (eg Kalinski), uncontrolled systemic inflammation (eg McMillan)... the list goes on.
SO my question is - (and yes, I can use Pubmed and read Nature Reviews as well as anyone) - for those of you who are research active in this field - experimentally and clinically - is a consensus emerging for the common solid tumours in particular?
* Does immune/inflammation support early tumour growth?
* Are patients immunologically tolerant to their tumours?
* Do inflammatory phenomena promote metastatic spread and/or seeding?
* How do the murine models - including immune reconstitution models - reflect human disease?
* What are the mistakes and deadends that get repeated with each new generation of researchers?
If there is sufficient interest and/or controversy in this area, then I'm going to commission a special issue of "Genes and Immunity" on the topic.
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* Does immune/inflammation support early tumour growth? Yes, I believe that the inflammatory response does promote early tumor growth.
* Are patients immunologically tolerant to their tumours? I believe that the immune system if capable of capturing most pre-cancerous cells and clearing them without the host even knowing. However, over time this selective pressure leads to "natural selection" of the most immune evasive tumor cells progressing to the next stage.
* Do inflammatory phenomena promote metastatic spread and/or seeding? I do believe that tumor cells secrete factors that promote the upregulation of chemokines at sites of metastasis.
* How do the murine models - including immune reconstitution models - reflect human disease? I think murine models hold alot of potential, if used correctly. I believe each murine model can be used to study and possibly translate knowledge with in a very narrow scope. Finding the right model for a specific question is important.
* What are the mistakes and dead ends that get repeated with each new generation of researchers? I am too early in my career to answer this.
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I wanted to test my cells ROS production by Reactive Oxygen Species (ROS) Detection Reagents (invitrogen), but I did not get positive results even with my control (PMA treated), ROS is lower than unstained cells. The reagent is not the problem because someone used before and got results.
The PMA treated I used is 50uM for 4 hours and while using flow cytometer, the living cells is enough for detection.
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I'm surprised you had any cells still alive (actually, they may be dead and this might explain your results). 50uM PMA is a lot, and 4 hours is quite long. With neutrophils and macrophages, 20-100nM PMA is sufficient to generate ROS within 10 seconds, and this is sustained for at least 10 minutes.
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